Background: Intraspecific variation in chromosome structure may cause genetic incompatibilities and thus provides the first step in the formation of species. In ants, chromosome number varies tremendously from 2n = 2 to 2n = 120, and several studies have revealed considerable variation in karyotype within species. However, most previous studies were limited to the description of chromosome number and morphology, and more detailed karyomorphometric analyses may reveal additional, substantial variation. Here, we studied karyotype length, genome size, and phylogeography of five populations of the fungus-farming ant Trachymyrmex holmgreni in order to detect potential barriers to gene flow. Results: Chromosome number and morphology did not vary among the five populations, but karyotype length and genome size were significantly higher in the southernmost populations than in the northern populations of this ant. Individuals or colonies with different karyotype lengths were not observed. Karyotype length variation appears to result from variation in centromere length. Conclusion: T. holmgreni shows considerable variation in karyotype length and might provide a second example of centromere drive in ants, similar to what has previously been observed in Solenopsis fire ants. Whether this variation leads to genetic incompatibilities between the different populations remains to be studied.